The overall goal of this project is to understand the biochemical events that enable a cell to tell 24-h time and temporally regulate cellular functions. A 24-h circadian clock is present in a wide range of species, from bacteria to humans, and disruptions in its underlying molecular mechanism can adversely affect fitness. Clock dysfunction in humans is related to a spectrum of health conditions such as cardiovascular disease, cancer, metabolic syndrome, mental illness, and sleep disorders. In cyanobacteria the fitness advantage conferred by a circadian clock whose periodicity resonates with the external day-night cycle has been demonstrated experimentally. A quantitative, systems-level, biochemical understanding is attainable for the circadian clock of the cyanobacterium Synechococcus elongatus, whose fundamental circadian oscillator can be reconstituted in vitro with three proteins, KaiA, KaiB, and KaiC. In this genetically tractable model organism it is possible to systematically alter the physical and biochemical properties of clock proteins and trace the impact of these changes from their proximal effects, through the protein-interaction network, to the expressed circadian phenotype. Discoveries in the previous funding period changed the view of the cyanobacterial clock from that of a 3-protein oscillator that communicates with input and output components to one of a larger interacting network in which the Kai proteins and additional components all contribute to timekeeping, phase resetting, and output signaling functions. This project will enhance the information that can be learned from the in vitro oscillator by expanding it into a fully-functional clock that exhibits output in the form f rhythmic phosphorylation of the transcription factor RpaA, recapitulates additional entrainment features through the input/output components CikA and SasA, and enables manipulation of competitive binding partners to assess their effects on period (Aim 1). Only metabolic effectors, rather than light transduction, have been identified thus far in providing environmental cues that entrain the clock to be synchronous with the light-dark cycle. The binding site, chemical identity, and spectral properties of a yellow cofactor associated with CikA will be determined, and engineered strains of S. elongatus that support metabolism of sugars in the dark will enable tests of light entrainment in a dark background (Aim 2). New evidence indicates that rhythmic expression of the genome, which is dependent on rhythms of phosphorylated RpaA, is also influenced by RpaB, whose phosphorylation is not controlled by the clock. Genetic and biochemical interaction experiments will determine the mechanism by which these two response regulators, not known to share the same kinase, influence each other's phosphorylation state, and whether they compete for binding at target promoters to generate circadian rhythms of gene expression (Aim 3). This project will provide the most comprehensive view available for the mechanisms that underlie circadian rhythmicity, and will provide insights into protein-protein interactions and signaling networks that are likely to operate in diverse bacteria, including pathogens.